The objective of the current proposal is to identify and characterize at a functional and structural level the proteins involved in heme transport in bacterial pathogens. Pathogenic bacteria require iron for their survival and growth and the ability to acquire this element is linked in part to their virulence. The major source of iron within the body is complexed in heme and heme-containing proteins. Pathogenic bacteria have developed sophisticated mechanisms by which they acquire iron from the hosts heme and hemeproteins. The current increase in the occurrence of antibiotic-resistant strains has created a need to develop alternative strategies to target such pathogens. Structural and functional characterization of proteins involved in heme transport and storage should make it possible to develop specific inhibitors. The expression and purification of the outer-membrane heme receptor (HutA) and periplasmic heme-binding protein (ShuT), will allow biochemical characterization of the heme transport proteins in gram-negative pathogens. The study will be directed towards two specific goals: first, the nature of heme binding to the receptor and transport proteins, and second, the mechanism of heme uptake. Site-directed mutagenesis together with physical techniques such as optical absorption, fluorescence, MCD and resonance Raman spectrophotometry will provide information on the structural features required for heme-binding and transport. The substrate-specificity of the outer-membrane receptor (HutA) and the periplasmic binding protein (ShuT) will be determined with substituted porphyrins, with the premise that such data may provide templates for the development of small molecule inhibitors. The expression of HutA in a heme deficient strain of E. coli allows us to directly monitor heme uptake as a function of cell viability and provides a direct method of determining heme transport in site-directed mutagenesis studies. In addition the coexpression of a bacterial heme oxygenase with the HutA receptor will be developed as a rapid colorimetric screen for future site-directed mutagenesis and inhibitor screening. The redox nature of heme transport will also be investigated with redox inactive non-iron metalloporphyrins such as Co and Zn-protoporphyrin IX. Once an understanding of the mechanism of heme transport in bacterial pathogens has been established the stage will be set for future design of selective inhibitors of heme transport.